Various 7-hydroxycoumarin-based compounds (also called umbelliferones) and their derivatives have been used as fluorescence indicators for enzymatic activity. The umbelliferones are fluorescent but when the 7-hydroxy group is functionalized with an enzyme-labile group, the fluorescence of the resulting compound is quenched. When contacted with an appropriate enzyme, the enzyme-labile group can be released through hydrolysis resulting in the reformation of the 7-hydroxy group and restoration of fluorescence.
Some known fluorescent indicators include those described, for example, in U.S. Pat. No. 5,830,912 (Gee et al.), U.S. Pat. No. 6,566,508 (Bentsen et al.), in the article by Gee et al., Analytical Biochemistry, 273, 41-48 (1999), and in the article by Guo et al, Huaxue Shiji, 32(8), 673-676 (2010).
One example fluorescent indicator is 4-methylumbelliferone glucuronide, which is referred to as 4-MUG. When exposed to a microorganism such as coliform, the non-fluorescent 4-MUG is transformed to the fluorescent compound 4-methylumbelliferone, which is referred to as 4-MU. When used as a fluorescent indicator for enzymatic activity, the pH of the sample typically must be basic to maximize the fluorescent signal of 4-MU. That is, compounds such as 4-MU can exist in both an acid and basic form with the basic form having significantly greater fluorescence at the wavelength of interest (e.g., the wavelength of maximum absorption, emission, or both).